A "cocktail"of numerous probe drugs to assess the metabolic activity of the corresponding cytochrome P450 enzymes requires that there is no problem of interaction among them. Some interactions among probe drugs can appear and may affect the rate of biotransformation of other ones. To develop a useful "cocktail" of probe drugs, our presented work was aimed on the influence of tolbutamide on cytochrome P450-mediated metabolism of bupropion. The biotransformation rates of bupropion administered either separately or in combined with tolbutamide were compared in this new study. The results revealed that tolbutamide had significantly decreased the rate of bupropion hydroxylation. Thus, due to shift in cytochrome P450 enzyme metabolic activity some extent differential results in the rate of P450-mediated metabolism can be observed when comparing assessment using combination of two model drugs with the common way (single marker use).